The present invention relates to a gene and a novel membrane protein encoded by said gene, and more specifically, relates to a gene encoding a novel membrane protein polypeptide having pre-B cell growth-supporting ability, a vector containing said gene, transformants transformed by said vector and a method for producing the novel membrane protein polypeptide by using said gene.
The present invention further relates to a monoclonal antibody recognizing a novel membrane protein polypeptide having pre-B cell growth-supporting ability.
The gene of the present invention encodes a novel membrane protein polypeptide enhancing pre-B cell growth-supporting ability on the surface of synovial cells derived from patients with rheumatoid arthritis (RA). In the present invention, a homogeneous and purified novel membrane protein polypeptide having pre-B cell growth-supporting ability can be produced in large quantities by transforming appropriate host cells with a suitable vector in which the gene of the present invention is inserted. Thus, according to the present invention, it becomes possible to identify rheumatoid arthritis (RA), and also prepare reagents for the clinical diagnosis thereof.
Inflammatory cells in the synovial membrane and the synovial fluid of patients with rheumatoid arthritis (RA) are derived from peripheral blood and the migration of these cells to synovial membranes has not been explicated perfectly yet, but it is believed to be caused by a complicated interaction between chemical signals given to cells and protein (adhesion molecule) on cell membranes.
Various studies upon the significance of membrane proteins in arthritis have been performed. For example, it is known that an intercellular adhesion molecule-1 (hereinafter referred to as ICAM-1) is expressed on the inner layer of the synovial membrane and the blood vessel of the synovial membrane of patients with rheumatoid arthritis (RA), which is a ligand of a T cell surface molecule LFA-1 and causes both adhesion and migration of cells in the blood vessel wall [Hale et al.; Arthritis Rheum., 32:22 (1989), and Haynes et al.; Springer Semin. Immunopathol., 11:163 (1989)]
Similarly, it is suggested that a vasocellular adhesion molecule-1 (hereinafter referred to as VCAM-1), which is a ligand of integrin VLA-4 expressed on T lymphoid cells (memory cells in particular) and monocytes, is expressed on the synovial membrane and fibroblast-like synovial cells of rheumatoid arthritis (RA) and osteoarthritis, [Morales-Ducret et al.; J. Immunol., 149:1424 (1992)], and further that a membrane protein called VAP-1 is expressed on the endothelial vein of a synovial membrane and may work as a specific recognition structure of leukocytes [Salmi et al.; Science, 257, 1407 (1992)].
The present inventors have engaged in extensive studies with a view to investigating the function of the bone marrow microenvironments in disorders causing abnormalities of B cells, and have found that the pre-B cell growth-supporting ability of bone marrow stromal cells derived from patients with rheumatoid arthritis (RA) and multiple myeloma (MM) is enhanced in comparison with that of healthy donor-derived bone marrow stromal cells and that the direct contact of pre-B cells with stromal cells might play an essential role in this supporting ability. And the present inventors have established novel stromal cell lines (RASV5-5, MMSV3-3) containing a molecule enhancing the growth of pre-B cells by cell-lining stromal cells of patients, and have found that the pre-B cell growth-supporting activity of these stromal cell lines is most likely caused by unknown adhesion molecules different from known stem cell factors (SCF), ICAM, CD44, VCAM-1, LFA-1xcex1, LFA-1xcex2, NCAM and FLAM-1 [J. Immunol., 149:4088 (1992)].
Further, since it has been suggested that the synovial cell line SynSV6-14 established from the synovial cell derived from patients with rheumatoid arthritis (RA) has pre-B cell growth-supporting ability similarly to the stromal cell line RASV5-5 derived from the bone marrow of patients with rheumatoid arthritis (RA), the present inventors have obtained a novel monoclonal antibody which responds to these cell lines but does not respond to the stromal cell line NFSV1-1 derived from the human bone marrow having no pre-B cell growth-supporting ability, and at the same time have succeeded in cloning genes encoding its antigen membrane protein (Bst-1) (Japanese Patent Application No. 5-141178/1993).
The present inventors have obtained a novel mouse monoclonal antibody RS38 which responds to SynSV6-14 but does not respond to the healthy bone marrow stromal cell line NFSV1-1 and recognizes a membrane protein different from the above Bst-1 at the process of producing various mouse monoclonal antibodies recognizing a membrane protein expressed on the synovial cell derived from patients with rheumatoid arthritis (RA) but not expressed on the cell derived from healthy donors. subsequently, the present inventors have succeeded in isolating clones encoding a novel membrane protein responding to said RS38, according to screening a cDNA library prepared from a synovial cell line derived from patients with rheumatoid arthritis (RA) by using the RS38 antibody, which has led to the completion of the present invention.
That is, the present invention is directed to provide a novel membrane protein polypeptide having pre-B cell growth-supporting ability, a gene encoding said polypeptide, a vector containing said gene, transformants transformed by said vector and a method for producing a novel membrane protein by using said gene.
Further, the present invention is directed to provide a monoclonal antibody recognizing a novel membrane protein having pre-B cell growth-supporting ability.
The present invention for accomplishing the above object consists of the following (1)-(7).
(1) A novel membrane protein polypeptide containing an amino acid sequence shown in sequence No. 1 of the sequence table or a part of the amino acid sequence and being expressed on the synovial membrane of patients with rheumatoid arthritis.
(2) A DNA encoding a polypeptide containing an amino acid sequence shown in sequence No. 1 of the sequence table or a part of the amino acid sequence.
(3) The DNA according to the above (2), characterized by containing a base sequence which hybridizes the base sequence shown in sequence No. 2 of the sequence table or a base sequence derived from said base sequence having at least one amino acid residue substituted, removed or added partially.
(4) A recombinant vector containing the DNA according to the above (2) or (3).
(5) A prokaryotic or eukaryotic host cell, characterized by being transformed with the recombinant vector according to the above (4).
(6) A method for producing the polypeptide containing an amino acid sequence shown in sequence No. 1 of the sequence table or a part of the amino acid sequence, characterized by culturing the host cell according to the above (5).
(7) A monoclonal antibody recognizing a polypeptide containing an amino acid sequence shown in sequence No. 1 of the sequence table or a part of the amino acid sequence.
Subsequently, the present invention will be described in detail.
The monoclonal antibody of the present invention may be prepared in the following manner essentially.
That is, the antibody of the present invention may be prepared by using a synovial cell derived from patients with rheumatoid arthritis (RA) having pre-B cell growth-supporting ability as an antigen, immunizing it according to an ordinary immunization method, cell-fusing the immunized cell according to an ordinary cell fusion method and cloning the fused cell according to an ordinary cloning method.
More specifically, as a preferable method for producing the monoclonal antibody of the present invention may be exemplified a method comprising using the cell line SynSV6-14, derived from the synovial membrane of patients with rheumatoid arthritis (RA) and established as a culture cell, as the above-mentioned antigen, fusing the plasma cell (immunocyte) of a mammal immunized with said antigen with a myeloma cell of a mammal such as a mouse, cloning the obtained fused cell (hybridoma), selecting clones producing the antibody of the present invention recognizing SynSV6-14 of them, and culturing them to recover the objective antibody.
In the method for producing the above monoclonal antibody, mammals to be immunized with the antigen are not particularly restricted; it is preferable to select one taking compatibility with a myeloma cell to be used for cell fusion into consideration and generally, a mouse, a rat and a hamster are used.
Immunization is performed according to a general method, for example, by administering cultured cells of the cell line SynSV6-14 derived from the synovial membrane of patients with rheumatoid arthritis (RA) into the peritoneal cavity of a mammal according to injection. More specifically, it is preferable to dilute it with or suspend it in PBS or physiological saline to a proper amount and administer it into an animal several times every 4-21 days, together with an ordinary adjuvant if required. In addition, an ordinary carrier (Schlepper) may be employed on the above administration. As an immunocyte, a splenic cell obtained after the final administration of the above cell line is used preferably.
As a myeloma cell of a mammal as the other parent cell to be fused with the above immunocyte may be preferably used known various cell lines including P3 (P3x63Ag8.653) [J. Immunol., 123:1548 (1978)], p3-U1 [Current Topics in Micro-biology and Immunology, 81:1-7 (1978)], NS-1 [Eur. J. Immumol., 6:511-519 (1976)], MPC-11 [Cell, 8:405-415 (1976)], SP2/0 [Nature, 276: 269-270 (1978)], FO [J. Immunol. Meth., 35:1-21 (1980)], S194 [J. Exp. Med., 148:313-323 (1978)] and R210 [Nature, 277:131-133 (1979)].
The cell fusion of the above immunocyte with a myeloma cell may be performed essentially according to a known method, for example, a method by Milstein et al. [Methods Enzymol., 73:3-46 (1981)].
More specifically, the above cell fusion may be performed, for example, in an ordinary nutrition medium in the presence of a fusion-accelerating agent. Examples of the fusion-accelerating agent include polyethylene glycol (PEG) and Sendai virus (HVJ), and moreover, auxiliary agents such as dimethyl sulfoxide may be added properly if required in order to enhance the fusing effect. Regarding the ratios of immunocytes and myeloma cells used, the former is preferably used in an amount 1-10 times that of the latter. Examples of a medium used in the above cell fusion include an RPMI-1640 medium and an MEM medium suitable for the proliferation of the above myeloma cell line and other mediums ordinarily used for the culture of this kind of cell, and in addition, supplementary serum such as fetal calf serum (FCS) may be used together.
Cell fusion is performed by mixing prescribed amounts of the above immunocytes and myeloma cells thoroughly in the above medium, adding a PEG solution preheated to about 37xc2x0 C., for example, PEG with an average molecular weight of the order of 1,000-6,000, to the medium ordinarily at a concentration of about 30-60% (W/V) and mixing them. Subsequently, by repeating the operations of adding proper mediums to them successively and centrifuging the reaction mixture, and removing the supernatants can be formed an objective hybridoma.
Said hybridoma is selected by culturing in an ordinary selective medium, for example, an HAT medium (medium containing hypoxanthine, aminopterin and thymidine). The culture in said HAT medium is continued for a time sufficient for cells other than objective hybridomas (non-fused cells) to die out, ordinarily for several days to several weeks. Subsequently, the screening and monocloning of the hybridomas producing the objective antibody are performed according to an ordinary limiting dilution analysis.
The thus prepared hybridomas producing the monoclonal antibody of the present invention may be subcultured in an ordinary medium and stored in liquid nitrogen for a long time.
In order to collect the monoclonal antibody of the present invention from said hybridomas may be employed a method comprising culturing said hybridomas according to an ordinary method and obtaining it from the supernatants or a method comprising administering a hybridoma into a appropriate mammal to proliferate and obtaining it from its ascites. The former is suitable for obtaining an antibody with a high purity and the latter is suitable for the mass production of the antibody.
Moreover, the antibody obtained according to the above method may be purified to have a high purity employing an ordinary purification means such as a salting out technique, gel filtration and affinity chromatography.
The thus prepared monoclonal antibody of the present invention makes it possible to identify synovial cells of patients with rheumatoid arthritis (RA) expressing a novel membrane protein of an antigen with a high sensitivity and a high precision according to an ordinary immunological means such as radioimmunoassay (RIA), enzyme immunoassay (EIA) and immunofluorescence analysis.
The gene of the present invention is obtained by preparing mRNA from a synovial cell of patients with rheumatoid arthritis (RA) expressing a membrane protein having human pre-B cell growth-supporting ability, and then converting it into a double-stranded cDNA according to a known method. As a cell used for preparing the mRNA can be mentioned, for example, a cell line SynSV6-14 used as an immune source of a hybridoma Rs38, but it is not limited to the cell line and therefore any type of cells expressing the membrane protein having human pre-B cell growth-supporting ability may be used. Incidentally, SynSv6-8 was used in the present invention.
For the preparation of the total RNA for obtaining mRNA can be employed a method for obtaining the total RNA which consists of performing cesium chloride density-gradient centrifugation after a guanidine thiocyanate treatment [Chirgwin et al., Biochemistry, 18:5294 (1979)], a method which consists of performing a surfactant treatment and a phenol treatment in the presence of the ribonuclease inhibitor of a vanadium complex [Berger and Birkenmeier, Biochemistry, 18:5143 (1979)], and other known methods.
The preparation of mRNA from the total RNA can be accomplished by recovering poly(A)+RNA from the total RNA according to, for example, affinity column chromatography using an oligo (dT)-bound carrier, for example, cephalose or cellulose, or a batch method. Besides, poly(A)+RNA can be further purified according to sucrose density-gradient centrifugation. In addition, there can be mentioned a method for obtaining poly(A)+RNA directly without preparing RNA or a convenient method using a commercially available kit.
In order to obtain a double-stranded cDNA from the thus obtained mRNA, for example, a DNA (cDNA) complementary to mRNA is synthesized by using mRNA as a template, and using an oligo (dT) complementary to a poly-A-chain sited at the 3xe2x80x2 end as a primer, and then treating it with reverse transcriptase.
The double-stranded cDNA can be also obtained by degrading mRNA according to an alkaline treatment, subjecting the obtained single-stranded cDNA as a template to a treatment with reverse transcriptase or DNA polymerase (e.g., Kienow fragment), and then treating it with SI nuclease, or treating it directly with RNase and DNA polymerase [Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory (1982) and Gubler and Hoffman, Gene, 25:263 (1983)]. Nowadays, convenient kits have been on the market, and a double-stranded cDNA can be obtained by using them.
The cDNA library can be obtained by inserting the thus obtained cDNA into a proper vector, for example, an EK-type plasmid vector such as pBR322 and pSC101, and a phage vector such as xcex gt10, and then transforming Escherichia coli with said vector (e.g., X1776, HB101, DH1, DH5) or the like (refer, for example, to xe2x80x9cMolecular Cloningxe2x80x9d above).
On the other hand, host cells of other prokaryotes and eukaryotes can be transformed by using a suitable expression vector in which the double-stranded cDNA obtained according to the above-mentioned method is inserted.
The ligation of the double-stranded cDNA to the vector can be performed by adding a proper chemically-synthesized DNA adapter thereto, and subjecting it with a vector DNA cleaved by means of a restriction enzyme in advance to a treatment with T4 phage DNA ligase in the presence of ATP.
The expression vector of the present invention contains a replicative origin, a selective marker, a promoter located in the upstream region of a gene to be expressed, an RNA splice site and a polyadenylated signal.
As a gene expression promoter in a mammal cell may be used virus promoters such as retrovirus, polyoma virus, adenovirus and simian virus (SV) 40, and promoters derived from cells such as human polypeptide chain elongation factor 1xcex1 (HEF-1xcex1). For example, in case of using a promoter of SV40, it can be performed easily according to a method of Mulligan et al. [Nature, 277:108 (1979)].
As a replicative origin can be used those derived from SV40 polyoma virus, adenovirus and bovine papilloma virus (BPV), and as a selective marker can be used a phosphotransferase APH (3xe2x80x2) II or I (neo) gene, a thymidine kinase (TK) gene, an Escherichia coli xanthine-guanine phosphoribosyl transferase (Ecogpt) gene and a dihydrofolate reductase (DHFR) gene.
In order to express the desired gene using a prokaryotic cell as a host cell, the host cell is transformed with a replicon derived from species capable of being fitted for hosts, namely, a plasmid vector containing a replicative origin and a regulation sequence. A vector which has a marker gene capable of imparting the selectivity of a phenotype to transformed cells is preferable. For example, in case of using Escherichia coli as a host cell, it can be transformed using pBR322, a vector originated from the host cell [Boliver et al., Gene, 2:95 (1975)]. The pBR322 contains an ampicillin resistant gene and a tetracycline resistant gene, and therefore transformants can be identified by utilizing either of these resistant properties.
As a promoter needed for the gene expression of a prokaryotic host cell can be mentioned a promoter of a xcex2-lactamase gene [Chang et al., Nature, 275:615 (1978)], a lactose promoter [Goeddle et al., Nature, 281:544 (1979)], a tryptophan promoter [Goeddle et al., Nucleic Acid Res., 8:4057 (1980)], a tac promoter and the like preferably; however, it is not limited to them.
As a prokaryotic host cell of hosts to be used in the expression system of the present invention can be mentioned Escherichia coli, Basillus subtilis, Bacillus thermophilus and the like preferably; however, it is not limited to them.
In addition, as an eukaryotic host cell can be mentioned eukaryotic microorganisms such as Saccharomyces cerevisiae, and cells derived from mammals such as a COS cell, a Chinese hamster ovary (CHO) cell, a C127 cell, a 3T3 cell, a Hela cell, a BHK cell, a namalwa cell and a human fetal renal cell (293 cell) preferably; however, it is not limited to them.
Incidentally, the culture of the transformants of the present invention may be performed by selecting culture conditions suitable for host cells appropriately.
The isolation of a cDNA encoding a membrane protein having pre-B cell growth-supporting ability of the present invention can be performed, for example, by using pre-B cell growth-supporting ability as an index or according to a method such as direct expression cloning using an antibody.
The measurement of pre-B cell growth-supporting ability can be performed by using a murine pre-B cell line DW34 [Eur. J. Immunol., 18:1767 (1988)]. That is, a cell expressing the membrane protein having pre-B growth-supporting ability is cultured until it becomes subconfluent on 24-well plates (preferable density being about 50%) and a proper amount of radiation is irradiated thereupon, DW34 of 1 to 2xc3x97103 per well is added thereto, and cultured in the RPMI-1640 medium containing 10% FCS under the condition of 5% CO2 at 37xc2x0 C. for about 4 to 6 days. The degree of the enhancement of the growth-supporting ability can be found by examining the number of viable cells of DW34 in each well according to trypan blue dye exclusion.
In the present invention, the desired gene could be cloned by repeating the steps, which consist of selecting a transformant expressing a membrane protein according to flow cytometry by means of an FACScan using a monoclonal antibody RS38 recognizing the novel membrane protein on the synovial cell of patients with rheumatoid arthritis (RA), preparing a transformant again by sorting the plasmid DNA used for the preparation of the transformant, and then screening the transformant according to flow cytometry.
Specifically, a transduced transformant (293T cell) was cultured on well plates and removed from the plates with PBS containing 0.02% EDTA, and after the cell was washed with an FACS buffer solution composed of PBS containing 2% FCS and 0.02% NaN3, it was reacted with RS38 as a primary antibody. Subsequently, after the unreacted primary antibody was removed by washing it with an FACS buffer solution, it was further reacted with a secondary antibody, an FITC-labeled antibody (FITC-labeled anti-mouse goat Ig antibody), dead cells were stained with propidium iodide, and viable cells were analyzed by an FACScan to select transformants responding strongly to RS38.
Further, the complete length of cDNA (pRS38-BOS) encoding a membrane protein polypeptide having novel pre-B cell growth-supporting ability shown in sequence No. 2 of the sequence table could be obtained by repeating the steps, which consist of treating Escherichia coli (DH5) containing the cDNA used for the preparation of transformants responding to the antibody with alkali to select a group of plasmids containing the desired gene, subdividing the group of plasmids into some groups of plasmids, transducing them into 293T cells again, and then selecting transformants according to FACScan analysis using the above-mentioned monoclonal antibody RS38.
Incidentally, the Escherichia coli DH5xcex1 strain containing pRS38-pUC19 with the cDNA inserted into the XbaI cleavage sites of a pUC19 vector was deposited at National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology in Japan, which is an international depositary authority according to Budapest Treaty on the international recognition of the deposit of microorganisms for the purpose of patent procedure, on Oct. 5, 1993, under the name of Escherichia Coli DH5xcex1 (pRS38-pUC19) with accession No. FERM BP-4434.
Generally, the genes of eukaryotes are thought to show polymorphism as known according to human interferon genes [e.g., Nishi et al., J. Biochem., 97: 153 (1985)], and in some cases at least one amino acid is substituted according to this polymorphism, and in other cases amino acids do not change at all though there are changes in the DNA sequence.
Further, it is probable that some polypeptides having at least one more or less amino acid than the amino acid sequence shown in sequence No. 1 of the sequence table, or some polypeptides substituted with at least one amino acid may also have the same function as that of the novel membrane protein of the present invention (pre-B cell growth-supporting ability). Actually, for example, it has been already known that the polypeptide obtained from a human interleukin-2 (IL-2) gene, in which a DNA sequence corresponding to cysteine is converted to a sequence corresponding to serine, also holds an IL-2 activity [Wang et al., Science, 224:1431 (1984)].
Moreover, a known protein gene and a gene shown in sequence No. 2 of the sequence table can be ligated by means of a proper restriction enzyme or adapter to yield a polypeptide bound to the known protein. As the known protein gene can be mentioned immunoglobulin, and it may be bound to a Fc portion thereof using the gene shown in sequence No. 2 of the sequence table instead of the variable region site thereof [(Zettlmeissl et al., DNA AND CELL BIOLOGY, 9:347-353 (1990)].
Furthermore, in case of expressing a polypeptide in eukaryotic cells, glycosylation occurs in many cases, and the glycosylation can be regulated according to the conversion of at least one amino acid; in this case, too, it may have the same function as that of the novel membrane protein polypeptide of the present invention. Therefore, even the genes in which the site encoding the membrane protein polypeptide of the present invention are modified artificially according to various methods as above and polypeptides can be included in the present invention so far as the polypeptides obtained from the genes have the same function as that of the membrane protein polypeptide of the present invention.
Moreover, it goes without saying that genes to be hybridized with genes shown in sequence No. 2 of the sequence table and polypeptides are also included in the present invention so far as the polypeptides expressed from the genes have the same function as that of the membrane protein polypeptide of the present invention (pre-B cell growth-supporting ability). In this case, hybridization may be carried out according to employing ordinary hybridization conditions (for example, refer to the above-mentioned xe2x80x9cMolecular Cloningxe2x80x9d).
The desired homogeneous and purified soluble membrane protein polypeptide having pre-B cell growth-supporting ability can be obtained by culturing a transformant transformed with a gene encoding the polypeptide, solubilizing the yielded polypeptide with a proper detergent, subjecting the resultant polypeptide to separation and purification. Preferable examples of the detergent include Nonidet P-40 (NP-40), Sodium Dodecyl Sulphate (SDS), Triton X-100, Tween 20 and the like.
In addition, soluble membrane proteins can be also prepared according to gene engineering. Namely, as shown in FIG. 3, since RS38 is guessed to be a cell membrane through-type protein having a cell membrane through domain and an intracellular domain at the side of the N terminal, soluble RS38 with the 49th Asn of sequence No. 1 of the sequence table as the N end can be prepared by employing a PCR-mutagenesis method [M. Kamman et al., Nucl. Acids Res., 15:5404 (1989)]. In this case, as a signal sequence may be used known ones and examples thereof include the signal sequence of Bst-1 (Japanese Patent Application No. 5-141178/1993) and that of G-CSF (Japanese Patent Publication No. 2-5395/1990).
As a means of separation and purification of the membrane protein polypeptide, a method to be used in the case of ordinary protein can be employed; for example, the membrane protein polypeptide of the present invention can be separated and purified properly by selecting and combining various types of chromatograpy such as affinity chromatography using the above-mentioned monoclonal antibody, ultrafiltration, salting out, dialysis and the like.